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Lodish Lab Research Summary
Research in my lab focuses on five important
areas at the interface between molecular cell biology
and medicine:
- Red blood cell development, especially on
the role of three signal transduction pathways downstream
of the erythropoietin receptor in controlling terminal
proliferation and differentiation of erythroid progenitor
cells;
- Hematopoietic stem cells, defining new cell
surface proteins for their purification and new growth
factors that support their expansion in culture;
- MicroRNAs, defining their roles in lineage
commitment of hematopoietic stem and progenitor cells,
and regulating muscle differentiation
- Adiponectin, a hormone we cloned that is
made exclusively by fat cells and that increases fatty
acid and glucose metabolism by muscle, and four homologous
proteins
- Regulated cleavage and release of the extracellular
domain ("ectodomain shedding") of transmembrane
precursors of several secreted growth factors.
Erythropoietin receptor (EpoR) and red cell
development: Epo and the EpoR are essential
for proliferation and differentiation of committed erythroid
progenitors, as is the cytosolic protein-tyrosine kinase
JAK-2. JAK2 binds to the EpoR cytosolic domain in the
endoplasmic reticulum and facilitates its folding to
promote cell surface expression. EpoRs exist on the
cell surface as inactive dimers; Epo binding changes
their conformation, leading to JAK2 transphosphorylation
and activation. JAK2 activates many signaling proteins
including PI-3’ kinase, the transcription factor
Stat5, and the Ras pathway. These pathways interact
to prevent apoptosis of committed erythroid progenitors
allowing them to undergo a predetermined program of
terminal proliferation and erythroid differentiation.
We showed that Stat5 directly activates transcription
of the anti-apoptotic protein bclxL. Stat5-/-mice exhibit
fetal anemia and increased apoptosis of erythroid progenitors
caused by reduced bclxL levels. Adult Stat5-/- mice
are anemic and deficient in generating high erythropoietic
rates in response to stress. Thus Stat5 controls one
rate-determining step regulating early erythroblast
survival. Activation of the PI-3’ kinase pathway
leads to activation of the Akt kinase and then phosphorylation
and inhibition of FOXO3a, a member of the Forkhead transcription
factor family. FOXO3a, in turn, activates transcription
of Tumor Necrosis Factor Apoptosis-Inducing Ligand (TRAIL).
We showed that inhibition of TRAIL production by Epo
addition partially rescues cells from apoptosis, demonstrating
the importance of this pathway in red cell formation.
By screening libraries of EpoRs with random mutations
in the transmembrane domain Xiaohui Lu identified several
point mutations that activate the EpoR in the absence
of ligand, including changes of either of the first
two transmembrane domains resides to cysteine. Xiaohui
then performed cysteine-scanning mutagenesis in the
EpoR juxtamembrane and transmembrane domains. Many mutants
formed disulfide-linked receptor dimers, but only EpoR
dimers linked by cysteines at three positions activated
EpoR signal transduction pathways and supported proliferation
of hematopoietic cells in the absence of cytokines.
These data suggest that activation of dimeric EpoR by
Epo binding is achieved by reorienting the EpoR transmembrane
and connected cytosolic domains and that certain disulfide-bonded
dimers represent the activated dimeric conformation
of the EpoR, constitutively activating downstream signaling.
Xiaohui is determining the structure of peptides corresponding
to these dimeric active a- helixes; this should shed
light on the structure of the Epo- activated receptor
transmembrane domain.
Little is known concerning the degradation of Epo in
the body – where this occurs or what may control
it. Alec Gross is studying the mechanism of Epo degradation,
both in erythroid cells expressing the EpoR and in mice
expressing abnormal numbers of Epo receptors in various
tissues. One goal is to explain why certain commercially-important
mutant Epo’s with extra carbohydrate chains have
a longer biological lifetime. Alec’s work using
cell lines showed that Epo degradation requires expression
of the EpoR. A fraction of the Epo bound to surface
receptors is internalized by endocytosis and degraded
in lysosomes. Most, however, either dissociates from
the surface receptor into the medium or is internalized
but resecreted. Long-lived mutant Epo binds slower and
dissociates more rapidly from surface Epo receptors,
but otherwise the kinetics of internalization, resecretion,
and degradation are indistinguishable from normal Epo.
As Alec’s kinetic modeling showed, these altered
receptor-binding kinetics can explain its longer half
life in vivo. To test this he will examine
the fate of Epo and its long-lived variants in mice
with altered numbers of Epo receptors in both hematopoietic
and non-hematopoietic cells; in this way he should discern
the role of surface EpoRs in normal Epo turnover.
Many of our current studies on EpoR signal transduction
make use of a new culture system Jing Zhang developed
where pure fetal liver erythroid progenitors (so-called
CFU-Es) undergo normal terminal proliferation and differentiation;
this can be followed on a cell-to-cell level by FACS.
As example, Jing showed that expression of a dominant-negative
H-ras in CFU-E progenitors, or addition of an inhibitor
of the MAP kinase pathway, did not affect erythroid
differentiation, indicating that activation of the Ras-
MAPK pathway by Epo is not essential for erythroid development.
To address the precise signaling pathway(s) regulated
by K-ras Jing then studied K-ras signaling in K-ras
-/- fetal liver erythroid progenitors. She
found that K-ras -/- fetal liver cells showed
a ~7-fold increase of apoptosis and significant delayed
erythroid differentiation. Moreover, when K-ras -/-
erythroid progenitors were cultured in vitro,
there is a significant delay in erythroid differentiation
but little increase in apoptosis. She then examined
the signaling pathways activated by Epo and stem cell
factor (SCF) in K-ras -/- fetal liver cells.
Epo- or SCF-dependent Akt activation was greatly reduced
in these cells whereas other pathways including Stat5
and p44/p42 MAP kinase were activated normally. Taken
together, her data identified K-ras as the major regulator
for cytokine-dependent Akt activation in erythropoiesis
in vivo.
Importantly, oncogenic mutations in ras genes
frequently occur in patients with myeloid disorders
and in these patients erythropoiesis is often affected.
Last year Jing showed that overexpression of oncogenic
H-ras in purified mouse primary fetal liver erythroid
progenitors blocks terminal erythroid differentiation
and supports Epo-independent proliferation. Jing showed
that three major pathways are abnormally activated by
oncogenic H-ras: Raf/ERK, PI3-kinase/Akt and RalGEF/RalA.
However, only constitutive activation of the MEK/ERK
pathway alone could recapitulate all of the effects
of oncogenic H-ras expression in blocking erythroid
differentiation and inducing Epo-independent proliferation.
Moreover, all effects of oncogenic H-ras expression
on primary erythroid cells were blocked by the addition
of a specific inhibitor of MEK1/2, allowing normal terminal
erythroid proliferation and differentiation. Jing’s
data suggest that the interruption of constitutive MEK/ERK
signaling is a potential therapeutic strategy to correct
impaired erythroid differentiation in patients with
myeloid disorders. But to avoid problems due to oncogenic
Ras overexpression, Jing, assisted by Yangang Liu, is
studying primary erythroid progenitors in which oncogenic
Ras is expressed from the endogenous Ras promoter. Expression
of oncogenic K-ras is induced using a rtTA/TetO-cre
system for a short period of time, and oncogenic K-ras
signaling will be assessed in highly purified primary
erythroid progenitors. Initial focus will be on the
signaling pathways constitutively activated by endogenous
oncogenic K-ras and hyperactivated in response to cytokine
stimulation. More importantly, the consequences of abnormal
oncogenic K-ras signaling in erythroid cells will be
evaluated at both the cellular and gene transcriptional
levels.
The cytosolic adaptor protein Lnk has been implicated
in cytokine receptor signaling. Recently Wei Tong, assisted
by Sara Zarnegar, showed that Lnk-deficient mice have
elevated numbers of erythroid progenitors, and that
splenic CFU-e progenitors are hypersensitive to Epo.
Lnk-/- mice also exhibit superior recovery after erythropoietic
stress. In addition, Lnk deficiency resulted in enhanced
Epo-induced signaling pathways in splenic erythroid
progenitors. Conversely, Lnk overexpression inhibits
Epo-induced cell proliferation. In primary culture of
fetal liver cells, Lnk overexpression inhibited Epo-dependent
erythroblast differentiation and induced apoptosis;
Lnk blocked all three major signaling pathways, Stat5,
Akt, and MAPK, induced by Epo in primary erythroblasts.
Wei showed that the Lnk SH2 domain is essential for
its inhibitory function, whereas the conserved tyrosine
near the C-terminus and the PH domain of Lnk are not
critical. Thus Lnk, through its SH2 domain, negatively
modulates EpoR signaling by attenuating JAK2 activation,
and regulates Epo-mediated erythropoiesis. Determining
the novel molecular mechanism by which Lnk inhibits
signaling from the EpoR and other cytokine receptors
is one of Wei’s current goals.
Another current project, conducted by Shilpa Hattangadi
and Jing Zhang, involves determining all changes in
gene expression that occur during terminal proliferation
and differentiation of purified fetal liver erythroid
cells. This involves assay of mRNAs by hybridization
to DNA gene microarrays (“gene chips)”.
Another, done by Shilpa in collaboration with members
of Rick Young’s
laboratory, involves immunoprecipitation of chromatin
with antibodies specific for transcription factors,
followed by hybridization of the recovered DNA to a
genomic DNA microarray. This protocol will enable Shilpa
to determine all of the genes that have critical erythroid-important
transcription factors bound to their promoter/enhancer
segments. Initial studies focus on transcriptional activation
by Stat5 but other factors will soon be investigated.
Shilpa’s long-term goal is to understand how the
complex pattern of gene expression during erythroid
development is controlled by transcription factors activated
by signal transduction pathways downstream of the EpoR.
A third project focuses on the role of integrins in
terminal proliferation and differentiation of purified
fetal liver erythroid cells, since adhesion of these
progenitors to fibronectin is essential for normal erythroid
development. Shawdee Eshghi showed that both α4β1 and
α5β1 integrins are present on erythroid progenitors,
and that α4β1 and α5β1 integrins support binding of
erythroid cells to different fibronectin domains. Shawdee
also showed that loss of both α4β1 and α5β1 integrins
during erythroid differentiation parallels the loss
of adhesion of erythroid cells to fibronectin. She is
now investigating the signal transduction pathways and
transcriptional changes mediated by each of these integrins.
She is also determining the pattern of integrin expression
on purified hematopoietic stem cells.
Epo prevents neuronal death during ischemic events
in the brain and in neurodegenerative diseases. The
molecular mechanisms of this protection are incompletely
understood. Using differentiated human neuroblastoma
cells Moon Um confirmed the antiapoptotic activity of
Epo and showed that Epo activates both the Stat5 and
PI-3 kinase/ AKT signaling pathways. Studies using expression
of chimeric mutant EpoRs able to activate neither or
only one of these pathways showed that activation of
both is required for EpoR activation to prevent neuronal
death. In parallel Moon is studying apoptosis of primary
adult neuronal cells genetically engineered to lack
the Epo receptor. Once she elucidates how Epo prevents
neuronal cell death in the brain, her findings could
lead to a novel clinical application of Epo for limiting
brain damage due to stroke or neurodegenerative diseases.
Joe Shuga, in collaboration with the laboratories of
Professors Leona Samson and Linda Griffith, is extending
our in vitro culture system for erythroid progenitors
into an assay for genotoxicity. Assays that predict
toxicity are an essential part of drug development and
many drugs fail in phase I clinical trials; therefore,
there is a demand for models that can better predict
human responses. The mouse in vivo micronucleus
(MN) assay is a robust toxicity test that assesses the
genotoxic effect of drugs by detecting chromosome fragments
that remain in the reticulocyte after enucleation; an
in vitro correlate to this assay might allow
extension to human cells and thus better predictive
power in drug development. As first steps in developing
a toxicity assay Joe is adapting our in vitro
erythropoiesis culture system to induce optimized erythropoietic
growth from Lin- populations from adult BM, and demonstrating
that exposure to genotoxicants induces MN-formation
in this culture system. In particular, Joe showed that
addition of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU)
to this culture system induces a global cytotoxic response
and concomitant decreases in erythropoietic differentiation
and increases in MN-formation. The increase in MN production
in the presence of BCNU provides a clear signal of the
clastogenic mechanism that likely induced the overall
hematopoietic toxicity.
Hematopoietic stem cells: Hematopoietic
stem cells (HSCs) are defined by their ability to self-renew
and to differentiate into all blood cell types. These
very rare cells form the basis of bone marrow transplantation
for treatment of leukemia and other cancers, and are
also a promising cell target for developing gene therapies
for treating a broad variety of human diseases. However,
development of these important clinical applications
of HSCs are greatly hampered by the lack of understanding
of the extracellular and intracellular signals that
govern their fates and the difficulty in ex vivo
expansion of these cells. We quantitate these cells
by bone marrow transplantation, monitoring the long-
term repopulation of the hematopoietic compartment of
lethally irradiated mice. This assay thus requires several
months to complete.
Several years ago Chang-Zheng Chen, a former fellow,
identified Endoglin, an ancillary TGF-β receptor,
as a surface marker for long-term repopulating mouse
bone marrow HSCs. He showed that bone marrow cells purified
by the EndoglinPositiveSca-1PositiveRhodamineLow
phenotype are a homogenous population of long- term
repopulating HSCs. Shawdee Eshghi recently showed that
these cells are morphologically homogenous and minute,
only ~ 5 – 7 μm in diameter. Thus the EndoglinPositiveSca-1PositiveRhodamineLow
phenotype defines a simple and effective procedure for
purifying a nearly homogenous stem cell population from
mouse bone marrow.
No single known growth factor or combination of growth
factors reproducibly supported HSC expansion in culture.
Furthermore existing lines of “supportive stromal
cells” did not support expansion of HSCs; at best
they maintained the level of HSCs over time, presumably
due to a steady state between generation of new HSCs
by division and differentiation of “old”
stem cells. Thus Chengcheng Zhang, assisted by Megan
Kaba, turned to mouse fetal liver since the number of
fetal HSCs normally increased markedly between embryonic
Day 12 and Day 16. Chengcheng hypothesized that unknown
growth proteins are produced by as-yet unidentified
populations of fetal liver cells that stimulate the
expansion of fetal liver HSCs. He then identified Embryonic
Day 15 fetal liver CD3+ Ter119-
cells as a completely novel cell population that supports
a net expansion of HSC numbers in culture. Although
CD3 is generally thought to be a specific T-cell marker,
these fetal liver CD3+ Ter119-
cells do not express other characteristic T cell markers.
By transcriptional profiling of these cells and several
others that do not support HSC expansion, Chengcheng
uncovered several novel growth factors that, together
supported an unprecedented extent of ex vivo expansion
of bone marrow HSCs. First he identified insulin-like
growth factor 2 (IGF - 2), which is specifically produced
by fetal liver CD3+ cells. Treatment of cocultures
of HSCs and day 15 fetal liver CD3+ Ter119-
cells with anti- IGF-2 antisera showed that IGF-2 is
a key molecule produced by these cells that stimulates
HSC expansion. Furthermore, when combined with other
growth factors IGF-2 is capable of markedly enhancing
ex vivo expansion of long-term repopulating fetal liver
and adult bone marrow HSCs. Systematic testing of combinations
of growth factors led to the development of a serum-free
culture medium containing low levels of SCF, TPO, IGF-2,
and FGF-1. As measured by competitive repopulation analyses,
there was a greater than 20-fold increase in numbers
of long-term HSCs after a 10-day culture of total BM
cells. Culture of a highly-enriched stem cell population,
for 10 days resulted in an ~8 fold expansion of repopulating
HSCs. Strikingly, the surface phenotype of ex vivo expanded
HSCs was different from that of freshly isolated HSCs,
but this plasticity of surface phenotype did not significantly
alter their repopulation capability.
More recently Chengcheng identified a novel and unstudied
protein specifically produced by day 15 fetal liver
CD3+ Ter119- cells that also stimulates
ex vivo expansion of HSCs. Chengcheng showed that, when
used in serum-free media in combination with other growth
factors, this protein stimulates a greater than 20-fold
expansion of HSCs following 10 days of culture of highly
enriched stem cells. Its receptor(s) and the signal
transduction pathway(s) it activates are unknown. A
main focus of Chengcheng’s current research is
deciphering the specific intracellular signal transduction
pathway(s) and transcriptional activations induced by
this protein in both cell lines and in HSCs.
Several years ago we identified 12 novel secreted and
cell surface proteins expressed specifically by lines
of stromal cells that support stem cell maintenance,
including several novel cytokines. During the past year
Alek Babic showed that one of these, pleiotrophin, also
supports ex vivo expansion of long- term repopulating
HSCs in culture. Currently Alek is investigating the
receptors for pleiotrophin in HSCs, as well as the signal
transduction pathways activated in these cells that
stimulate HSC expansion.
Among the other proteins specifically expressed by
Day 15 fetal liver CD3+ cells was the prion
protein (PrP), a glycosylphosphatidylinositol (GPI)-
anchored cell surface protein; despite many years of
research the normal function of PrP was unknown. Chengcheng
Zhang surmised that PrP would also be expressed on long-term
repopulating hematopoietic stem cells and initiated
a collaboration with Professor Susan
Lindquist and her PhD student Andrew Steele. Not
only did they quickly confirm this hypothesis, they
went on to show that HSCs from PrP-/- bone
marrow exhibit impaired activities in serial transplantation
experiments. Most strikingly, ectopic expression of
PrP in PrP-/- bone marrow cells rescued the
defects in hematopoietic engraftment. Therefore, PrP
is a novel marker for HSCs and supports their self-renewal
during successive bone marrow transplantations. Together
with Andrew, Chengcheng is trying to determine the molecular
function of PrP. PrP might be the coreceptor for a hormone
affecting HSC activity, possibly concentrating this
as yet unidentified molecule on the cell surface and/or
presenting it to the signaling receptor(s). Alternatively,
PrP might interact with proteins in the BM extracellular
matrix or on the surface of stromal cells, and possibly
support retention of transplanted HSCs within the BM
microenvironment.
MicroRNAs that modulate differentiation: MicroRNAs
(miRNAs) are ~22-nt non-coding RNAs that can play important
roles in development by targeting the messages of protein-coding
genes for cleavage or repression of productive translation.
Examples include lin-4 and let-7 miRNAs
that control the timing of C. elegans larval
development. As shown by the Bartel
laboratory and others, humans have between 250 and 300
genes that encode miRNAs, an abundance corresponding
to almost one percent of protein-coding genes. Based
on the evolutionary conservation of many miRNAs among
different animal lineages, it is reasonable to suspect
that some mammalian miRNAs might also have important
functions during development.
As a first step towards testing the idea that miRNAs
might play roles in mammalian development, and more
specifically hematopoiesis, Chang-Zheng Chen, in collaboration
with Prof. David Bartel, cloned about 100 unique miRNAs
from mouse bone marrow. Three, miR-181, miR-223, and
miR-142s, were exclusively or preferentially expressed
in hematopoietic tissues. miR-181 was very strongly
expressed in thymus, the primary lymphoid organ, which
mainly contains T-lymphocytes. Mature miR-181 expression
in the bone marrow cells was detectable in undifferentiated
Lin- progenitor cells and up-regulated in differentiated
B-lymphocytes, marked by the B220 surface antigen. In
other differentiated lineages, miR-181 expression did
not increase over that seen in Lin- cells. Using retrovirus
vectors he developed, Chang- Zheng ectopically expressed
miR-181 in a population of bone marrow hematopoietic
stem and progenitor cells. This led to an increased
fraction of B-lineage cells both in tissue-culture differentiation
assays and in transplanted adult mice; there was a corresponding
decrease in CD-8+ T cells. Expression of
miR-142s, in contrast, was most abundant in cells of
the granulocyte and macrophage lineages. Overexpression
of miR-142s in hematopoietic stem and progenitor cells
led to an increase in the numbers of granulocytes and
macrophages and a decrease in numbers of both mature
CD-8+ and CD-4+ T cells. These
results indicate that microRNAs are components of the
molecular circuitry controlling mouse hematopoiesis
and suggest that other microRNAs have similar regulatory
roles during other facets of vertebrate development.
Current projects aim to uncover the mRNAs downregulated
by miR-181 and miR-142s, and much of this work is being
done in Chang-Zheng’s new laboratory at the Stanford
University School of Medicine.
Beiyan Zhou has used microRNA microarrays developed
in the Bartel laboratory to identify several miRNAs
specifically upregulated in populations of thymic and
splenic hematopoietic cells she isolated: B cells, immature
CD-4- CD-8- and CD-4+
CD-8+ T cells, as well as in more mature
thymic CD-4- CD-8+ and CD-4+
CD-8- T cells. She is confirming these results
by Northern blotting. Overexpression of these miRNAs
in cell lines and animals, as well as “knocking
down” their expression in cultured cells, should
shed light on the roles of these miRNAs in B- and T-
cell development as well as in hematopoiesis more broadly.
Prakash Rao has identified three evolutionarily conserved
muscle- specific several miRNAs that are upregulated
during differentiation of cultured C2C12 myoblasts into
differentiated myotubes. Prakash and Mina Farkhondeh,
a UROP student, are collaborating with members of the
Bartel laboratory to determine the direct targets of
these miRNAs. They are also using several new technologies
in an attempt to “knock down” expression
of these miRNAs and thus learn more about their specific
roles in muscle differentiation. Prakash is also working
with Guangtao Ge, a former lab member, to predict the
sequences upstream of these muscle- specific miRNA genes
that regulate their transcription. They have identified
several such sites and they are currently using multiple
experimental approaches to determine whether these indeed
regulate miRNA expression.
I-hung Shih, a postdoctoral fellow in the Bartel lab
who has been working closely with us, has identified
several micro RNAs that are upregulated during differentiation
in culture of 3T3- L1 preadipocytes to adipocytes. I-hung
is currently examining the effects on adipocyte differentiation
of overexpressing these micro RNAs in preadipocytes,
and knocking down their expression during adipogenesis.
Using computational and experimental techniques developed
in the Bartel lab she is trying to determine the mRNA
target(s) of these miRNAs.
Adiponectin and its homologs: In 1995
we cloned adiponectin, originally called Acrp30, as
novel adipocyte-specific secreted protein hormone. Adiponectin
addition potently elevates fat and glucose catabolism
by muscle, enhances glycogen accumulation in muscle,
and inhibits gluconeogenesis in liver. Mutations in
the adiponectin gene are linked to development of adult-onset
diabetes and the levels of adiponectin in serum are
reduced in obese and diabetic patients and mice. Circulating
adiponectin levels negatively correlated with human
plasma triglyceride and fasting insulin levels and several
clinical studies showed those with low adiponectin levels
are more likely to develop type II diabetes mellitus
and cardiovascular disease. This data suggests that
adiponectin is a potential genetic determinant of insulin
sensitivity.
Adiponectin has four domains: a cleaved amino-terminal
signal sequence, a region without homology to known
proteins, a collagen-like region, and a globular segment
at the carboxy-terminus. The three-dimensional structure
of the globular domain is strikingly similar to that
of TNF-α even though there is no homology at the
primary sequence level. Like TNF-α the globular
domain forms homotrimers, and intermolecular disulfide
bonds generate hexameric and high molecular weight Adiponectin
species.
In collaboration with the Ruderman laboratory at B.U.
Medical School, Tsu-Shuen Tsao showed that treatment
of rat striated muscle with trimeric adiponectin led
to phosphorylation and activation of AMP-activated protein
kinase (AMPK), an enzyme that when activated causes
increases in muscle fatty acid oxidation, glucose uptake
and oxidation, and insulin sensitivity. Adiponectin-
mediated activation of AMPK caused phosphorylation and
thus diminished activity of acetyl CoA carboxylase,
a corresponding decrease in the concentration of malonyl
CoA, and a corresponding increase in long-chain fatty
acid oxidation. In addition, adiponectin caused an increase
in glucose uptake. Both in vivo and in muscle
culture adiponectin most likely exerts its actions on
muscle fatty acid oxidation by inactivating ACC, via
activation of AMPK and perhaps other signal transduction
proteins.
AMPK is composed of three subunits – the a kinase
subunit that undergoes regulated phosphorylation, the
γ subunit that binds AMP, and the β subunit
that is thought to act as a scaffold that binds to both
the α and γ subunits. Cellular and physiological
stresses that deplete ATP such as nutrient deprivation,
hypoxia, ischemia, and exercise in muscle all lead to
activation of AMPK. Kelly Wong has been redetermining
the interactions of the three AMPK subunits and has
shown that several key aspects of the current model
are wrong. Most significantly, Kelly showed that the
α-subunit binds directly to the γ-subunit,
in striking contradiction to the “standard”
model. He also showed that the “scaffolding”
β-subunit does not bind directly to the γ-subunit;
interactions of the β- and γ- subunits can
be detected only if the α-subunit is also present.
Thus his data suggests a model for AMPK structure in
which the β-and the γ-subunit bind directly to the α-subunit,
and in which the β-subunit does not bind directly to
the AMP-sensing γ-subunit. Kelly is currently determining
the precise subunit composition of intermediates in
AMPK assembly and deciphering the molecular mechanism
by which AMP binding to the γ-subunit allosterically
activates the α-subunit kinase. He also aims to determine
whether all three AMPK subunits are essential for kinase
activation by adiponectin; in the process he hopes to
identify other proteins that might connect the AMPK
α-subunit to the elusive signaling adiponectin receptors.
Tsu-Shuen Tsao and Christopher Hug assessed if adiponectin
is a signaling molecule by searching for promoter or
enhancer elements that respond to hormone addition.
Addition of hexameric and larger isoforms of adiponectin
to C2C12 myocytes or myotubes leads to activation of
NF-κB transcription factor in a manner dependent upon
phosphorylation and degradation of the IκB-α subunit;
trimeric adiponectin has no effect. In contrast, only
trimeric adiponectin but not hexameric and larger isoforms,
activates AMPK in muscle. Their data indicates that
oligomerization of adiponectin is important for at least
some of its biological activities, and that changes
in the relative abundance of each oligomeric isoform
in plasma may regulate adiponectin activity. These results
also suggest that hexameric and trimeric adiponectin
might bind to different receptors and/or activate different
intracellular signal transduction pathways.
Christopher Hug, assisted by Jin Wang, used an expression
cloning strategy to identify T-cadherin as a receptor
for hexameric and high molecular weight forms of adiponectin.
T-cadherin is highly and specifically expressed in the
vasculature, where it is predominantly found in endothelial
and smooth muscle cells in the blood vessel intima.
T-cadherin is attached to the membrane via a GPI anchor
at the C-terminus. Chris’ preliminary studies
indicate that it is the major adiponectin binding protein
in the body, as deletion of T-cadherin results in a
many-fold increase in the level of high molecular weight
adiponectin in the circulation. T- cadherin is upregulated
following vascular injury and he hypothesizes that,
by binding to adiponectin, it may play a role in atherosclerosis
progression. Adiponectin also binds to extracellular
collagens exposed during vessel wall injury; adiponectin
also contains a KGD sequence that we hypothesize to
bind to integrins, a point that will be examined by
testing whether or not thrombosis is affected in adiponectin
-/- mice. Currently Chris is determining the role of
T-cadherin in adiponectin activation of the AMPK and
NF-κB signal transduction pathways. Chris and Jin are
also cloning other cell surface adiponectin receptors
including those that directly activate AMPK.
Guang Wong, with the assistance of Sarah Krawczyk,
used multiple genomic approaches to identify a family
of seven highly conserved human and mouse proteins homologous
in sequence and presumed structure to adiponectin. These
are designated as C1q/TNF-a related proteins (CTRP)-1
to 7 Expression of CTRP1, 2, 3, and 7 mRNAs, like that
of adiponectin, is far higher in adipose tissue that
in any other tissue tested. Like that of adiponectin,
expression of CTRP1, 2, 3, and 7 mRNAs in 3T3- L1 adipocytes
is upregulated by treatment with a thiazolidinedione
agonist of PPAR-g. CTRP2 is the closest paralog of adiponectin;
Guang’s data show that CTRP2 is structurally homologous
to adiponectin in that both form higher order structures
including trimers and hexamers. Moreover, CTRP1, 2,
3, and 7 are functionally homologous to adiponectin
in their ability to activate the key metabolic sensor
AMP-activated protein kinase (AMPK) in muscle and lung
cells. Similar to adiponectin, treatment of C2C12 myotubes
with CTRP2 (the others have not yet been tested) resulted
in increased accumulation of glycogen and enhanced oxidation
of long chain fatty acids, the latter due to phosphorylation
of Acetyl CoA carboxylase (ACC) by AMPK. Taken together,
these results suggest significant metabolic functions
for CTRP1, 2, 3, and 7, but the natural target cells
of these hormones and the functions they control are
not known. An understanding of the natural metabolic
functions of these hormones will likely emerge from
analysis of the CTRP- overexpressing transgenic mice
and CTRP gene knock- out mice Guang is now generating.
Guang is also using expression cloning strategies to
identify the receptors for these novel proteins. This
discovery of a family of adiponectin paralogs has implications
for understanding the control of energy homeostasis
and could provide new targets for pharmacologic intervention
in metabolic diseases such as diabetes and obesity.
Regulated cleavage and release of the extracellular
domain of transmembrane precursors of several secreted
growth factors. Protease cleavage and release
of the extracellular domain (ECD, "ectodomain shedding")
of a multitude of transmembrane proteins has been linked
to the activation of many signaling pathways including
the MAPK pathway. Cleavage of the ECD is mostly carried
out by metalloproteases (MMPs) of the ADAM family (“a
disintegrin and metalloprotease”). ECD cleavage
is often followed by and is a prerequisite for intramembranous
cleavage of the intracellular domain (ICD) of the same
protein by ?-secretase; some of the cleaved ICDs translocate
to the nucleus, where they may regulate gene transcription.
Membrane-spanning pro-hormone ligands of the epidermal
growth factor receptor (HER) family are well studied
examples of proteins that undergo ectodomain shedding
and are physiologically important in many cellular contexts
in organisms from Drosophila to mammals. But how the
ectodomain cleavage machinery is regulated is largely
unknown, as only a few specific stimuli which induce
ectodomain shedding have been identified. Activation
of the cardiac β-adrenergic receptor leads to HB-EGF-cleavage-mediated
development of cardiac hypertrophy. Andreas Herrlich
showed that another HER-ligand, neuregulin1β (NRG1β),
is cleaved by an MMP in response to hypertonic stress
and subsequently activates EGF-family receptors in an
autocrine fashion. This signaling step leads to MAPK
activation followed by enhanced expression of genes
encoding water channels (aquaporins). Regulation of
ectodomain cleavage could occur at least two levels
- at the level of the MMP or via covalent modifications
of the target protein, such as phosphorylation or ubiquitination
on the cytosolic face.
Andreas, and Cameron Sadegh, a UROP student, are cloning
novel genes that regulate ectodomain shedding using
a high-throughput expression cloning strategy. They
can detect cleavage of all chosen HER-ligands by either
hypertonic stress, phorbol ester addition, or stimulation
with lysophosphatidic acid in a FACS-based assay using
mouse lung epithelial (MLE) cell clones stably expressing
one of the chosen pro-hormone ligands. The ligands are
tagged at the extracellular domain with one of several
epitope tags; at its cytosol-facing terminus the proteins
have been fused with EGFP. The extracellular epitope
of the transmembrane pro-hormone ligand is detected
with a fluorochrome-coupled (red) anti-epitope antibody,
while the intracellular domain EGFP-fusion is detected
by green fluorescence. Stimulation of cleavage results
in a decrease of the red to green fluorescence ratio,
while inhibition of basal or induced cleavage is reflected
by an increase in this ratio. Reporter cell clones have
been infected with a retroviral library generated from
a cleavage competent cell line and cells that exhibit
altered cleavage of the doubly tagged HER ligands. are
being sorted and cloned. Genomic PCR followed by cloning
will allow detection of the particular cDNA library
insert in the isolated cell clones that encodes a protein
that either activates or inhibits regulated ectodomain
shedding. This protocol should enable Andreasand Cameron
to identify and clone novel proteins that regulate shedding
of the ectodomain of members of the EGF family of hormones.
Hideshiro Saito-Benz will be using similar technologies
to identify novel proteins that regulate cleavage of
APP, a transmembrane protein whose cleavage products
are thought to precipitate certain cases of Alzheimer’s
disease.
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